Actin remodeling by ADF/cofilin is required for cargo sorting at the trans-Golgi network

Knockdown of the actin-severing protein actin-depolymerizing factor (ADF)/cofilin inhibited export of an exogenously expressed soluble secretory protein from Golgi membranes in Drosophila melanogaster and mammalian tissue culture cells. A stable isotope labeling by amino acids in cell culture mass spectrometry–based protein profiling revealed that a large number of endogenous secretory proteins in mammalian cells were not secreted upon ADF/cofilin knockdown. Although many secretory proteins were retained, a Golgi-resident protein and a lysosomal hydrolase were aberrantly secreted upon ADF/cofilin knockdown. Overall, our findings indicate that inactivation of ADF/cofilin perturbed the sorting of a subset of both soluble and integral membrane proteins at the trans-Golgi network (TGN). We suggest that ADF/cofilin-dependent actin trimming generates a sorting domain at the TGN, which filters secretory cargo for export, and that uncontrolled growth of this domain causes missorting of proteins. This type of actin-dependent compartmentalization and filtering of secretory cargo at the TGN by ADF/cofilin could explain sorting of proteins that are destined to the cell surface.     

Solution structure of human cofilin: actin binding, pH sensitivity, and relationship to actin-depolymerizing factor

Human actin-depolymerizing factor (ADF) and cofilin are pH-sensitive, actin-depolymerizing proteins. Although 72% identical in sequence, ADF has a much higher depolymerizing activity than cofilin at pH 8. To understand this, we solved the structure of human cofilin using nuclear magnetic resonance and compared it with human ADF. Important sequence differences between vertebrate ADF/cofilins were correlated with unique structural determinants in the F-actin-binding site to account for differences in biochemical activities of the two proteins. Cofilin has a short beta-strand at the C terminus, not found in ADF, which packs against strands beta3/beta4, changing the environment around Lys96, a residue essential for F-actin binding. A salt bridge involving His133 and Asp98 (Glu98 in ADF) may explain the pH sensitivity of human cofilin and ADF; these two residues are fully conserved in vertebrate ADF/cofilins. Chemical shift perturbations identified residues that (i) differ in their chemical environments between wild type cofilin and mutants S3D, which has greatly reduced G-actin binding, and K96Q, which does not bind F-actin; (ii) are affected when G-actin binds cofilin; and (iii) are affected by pH change from 6 to 8. Many residues affected by G-actin binding also show perturbation in the mutants or in response to pH. Our evidence suggests the involvement of residues 133-138 of strand beta5 in all of the activities examined. Because residues in beta5 are perturbed by mutations that affect both G-actin and F-actin binding, this strand forms a "boundary" or "bridge" between the proposed F- and G-actin-binding sites.     

Regulation of actin filament dynamics by actin depolymerizing factor/cofilin and actin-interacting protein 1: new blades for twisted filaments

Actin depolymerizing factor (ADF)/cofilin enhances turnover of actin filaments by severing and depolymerizing filaments. A number of proteins functionally interact with ADF/cofilin to modulate the dynamics of actin filaments. Actin-interacting protein 1 (AIP1) has emerged as a conserved WD-repeat protein that specifically enhances ADF/cofilin-induced actin dynamics. Interaction of AIP1 with actin was originally characterized by a yeast two-hybrid system. However, biochemical studies revealed its unique activity on ADF/cofilin-bound actin filaments. AIP1 alone has negligible effects on actin filament dynamics, whereas in the presence of ADF/cofilin, AIP1 enhances filament fragmentation by capping ends of severed filaments. Studies in model organisms demonstrated that AIP1 genetically interacts with ADF/cofilin and participates in several actin-dependent cellular events. The crystal structure of AIP1 revealed its unique structure with two seven-bladed beta-propeller domains. Thus, AIP1 is a new class of actin regulatory proteins that selectively enhances ADF/cofilin-dependent actin filament dynamics.     

ADF/cofilin regulates actomyosin assembly through competitive inhibition of myosin II binding to F-actin

The contractile actin cortex is important for diverse fundamental cell processes, but little is known about how the assembly of F-actin and myosin II motors is regulated. We report that depletion of actin depolymerizing factor (ADF)/cofilin proteins in human cells causes increased contractile cortical actomyosin assembly. Remarkably, our data reveal that the major cellular defects resulting from ADF/cofilin depletion, including cortical F-actin accumulation, were largely due to excessive myosin II activity. We identify that ADF/cofilins from unicellular organisms to humans share a conserved activity to inhibit myosin II binding to F-actin, indicating a mechanistic rationale for our cellular results. Our study establishes an essential requirement for ADF/cofilin proteins in the control of normal cortical contractility and in processes such as mitotic karyokinesis. We propose that ADF/cofilin proteins are necessary for controlling actomyosin assembly and intracellular contractile force generation, a function of equal physiological importance to their established roles in mediating F-actin turnover.     

Efficient Salmonella entry requires activity cycles of host ADF and cofilin

Entry of Salmonella into mammalian cells is strictly dependent on the reorganization of actin cytoskeleton induced by a panel of Salmonella type III secreted proteins. Although several factors have been identified to be responsible for inducing the actin polymerization and stability, little is known about how the actin depolymerization contributes to Salmonella-induced actin rearrangements. We report here that activity cycles of host actin depolymerizing factor (ADF and cofilin) are modulated by Salmonella during bacterial entry. Efficient Salmonella internalization involves an initial dephosphorylation of ADF and cofilin followed by phosphorylation, suggesting that ADF and cofilin activities are increased briefly. Expression of a kinase dead form of an ADF/cofilin kinase (LIM kinase 1) or a catalytically inactive ADF/cofilin phosphatase (Slingshot), but not constitutively active LIM kinase 1 or wild-type Slingshot, resulted in decreased invasion. These data suggest that ADF/cofilin activities play a key role in the actin polymerization/depolymerization process induced by Salmonella. The activation of ADF/cofilin is brief and has to be reversed to facilitate efficient bacterial entry. Surprisingly, co-expression of constitutive active ADF and cofilin prevented efficient Salmonella entry, whereas expression of either one alone had no effect. We propose that ADF and cofilin actin-dynamizing activities and their activity cycling via phosphorylation are required for efficient Salmonella internalization.     

Sequence of cDNAs encoding actin depolymerizing factor and cofilin of embryonic chicken skeletal muscle: two functionally distinct actin-regulatory proteins exhibit high structural homology.

Two actin-regulatory proteins of 19 and 20 kDa are involved in the regulation of actin assembly in developing chicken skeletal muscle. They are homologous with actin depolymerizing factor (ADF) and cofilin, a pH-dependent actin-modulating protein, which were originally discovered in chicken and mammalian brain, respectively. In this study, full-length cDNA clones were isolated by screening a lambda gt11 cDNA library constructed from poly(A+) RNA of embryonic chicken skeletal muscle with the antibodies specific for each protein, and their complete sequences were determined. The chicken cofilin cDNA encoded a protein of 166 amino acids, the sequence of which had over 80% identity with that of porcine brain cofilin. The amino acid sequence of the ADF was 165 amino acids and showed about 70% identity with either chicken or mammalian cofilin, in spite of the fact that ADF and cofilin are functionally distinct. Like chicken and mammalian cofilin, ADF contained a sequence similar to the nuclear transport signal sequence of SV40 large T antigen. ADF and cofilin shared a hexapeptide identical with the amino-terminal sequence of tropomyosin as well as the regions homologous to other actin-regulatory proteins, including depactin, gelsolin, and profilin. The overall nucleotide sequences and Southern blot analysis of genomic DNA, however, indicated that the two proteins were derived from different genes.     

Actin depolymerization factor/cofilin activation regulates actin polymerization and tension development in canine tracheal smooth muscle

The contractile activation of airway smooth muscle tissues stimulates actin polymerization, and the inhibition of actin polymerization inhibits tension development. Actin-depolymerizing factor (ADF) and cofilin are members of a family of actin-binding proteins that mediate the severing of F-actin when activated by dephosphorylation at serine 3. The role of ADF/cofilin activation in the regulation of actin dynamics and tension development during the contractile activation of smooth muscle was evaluated in intact canine tracheal smooth muscle tissues. Two-dimensional gel electrophoresis revealed that ADF and cofilin exist in similar proportions in the muscle tissues, and that approximately 40% of the total ADF/cofilin in unstimulated tissues is phosphorylated. Phospho-ADF/cofilin decreased concurrently with tension development in response to stimulation with acetylcholine (ACh) or potassium depolarization indicating the activation of ADF/cofilin. Expression of an inactive phospho-cofilin mimetic (cofilin S3E) but not wild type cofilin in the smooth muscle tissues inhibited endogenous ADF/cofilin dephosphorylation and ACh-induced actin polymerization. Expression of cofilin S3E in the tissues depressed tension development in response to ACh, but it did not affect myosin light chain phosphorylation. The ACh-induced dephosphorylation of ADF/cofilin required the Ca2+-dependent activation of calcineurin (PP2B). The results indicate that the activation of ADF/cofilin is regulated by contractile stimulation in tracheal smooth muscle and that cofilin activation is required for actin polymerization and tension development in response to contractile stimulation.     

Mapping the ADF/cofilin binding site on monomeric actin by competitive cross-linking and peptide array: evidence for a second binding site on monomeric actin

The binding sites for actin depolymerising factor (ADF) and cofilin on G-actin have been mapped by competitive chemical cross-linking using deoxyribonuclease I (DNase I), gelsolin segment 1 (G1), thymosin beta4 (Tbeta4), and vitamin D-binding protein (DbP). To reduce ADF/cofilin induced actin oligomerisation we used ADP-ribosylated actin. Both vitamin D-binding protein and thymosin beta4 inhibit binding by ADF or cofilin, while cofilin or ADF and DNase I bind simultaneously. Competition was observed between ADF or cofilin and G1, supporting the hypothesis that cofilin preferentially binds in the cleft between sub-domains 1 and 3, similar to or overlapping the binding site of G1. Because the affinity of G1 is much higher than that of ADF or cofilin, even at a 20-fold excess of the latter, the complexes contained predominantly G1. Nevertheless, cross-linking studies using actin:G1 complexes and ADF or cofilin showed the presence of low concentrations of ternary complexes containing both ADF or cofilin and G1. Thus, even with monomeric actin, it is shown for the first time that binding sites for both G1 and ADF or cofilin can be occupied simultaneously, confirming the existence of two separate binding sites. Employing a peptide array with overlapping sequences of actin overlaid by cofilin, we have identified five sequence stretches of actin able to bind cofilin. These sequences are located within the regions of F-actin predicted to bind cofilin in the model derived from image reconstructions of electron microscopical images of cofilin-decorated filaments. Three of the peptides map to the cleft region between sub-domains 1 and 3 of the upper actin along the two-start long-pitch helix, while the other two are in the DNase I loop corresponding to the site of the lower actin in the helix. In the absence of any crystal structures of ADF or cofilin in complex with actin, these studies provide further information about the binding sites on F-actin for these important actin regulatory proteins.     

Microscopic evidence that actin-interacting protein 1 actively disassembles actin-depolymerizing factor/Cofilin-bound actin filaments

Actin-depolymerizing factor (ADF)/cofilin and gelsolin are the two major factors to enhance actin filament disassembly. Actin-interacting protein 1 (AIP1) enhances fragmentation of ADF/cofilin-bound filaments and caps the barbed ends. However, the mechanism by which AIP1 disassembles ADF/cofilin-bound filaments is not clearly understood. Here, we directly observed the effects of these proteins on filamentous actin by fluorescence microscopy and gained novel insight into the function of ADF/cofilin and AIP1. ADF/cofilin severed filaments and AIP1 strongly enhanced disassembly at nanomolar concentrations. However, gelsolin, gelsolin-actin complex, or cytochalasin D did not enhance disassembly by ADF/cofilin, suggesting that the strong activity of AIP1 cannot be explained by simple barbed end capping. Barbed end capping by ADF/cofilin and AIP1 was weak and allowed filament elongation, whereas gelsolin or gelsolin-actin complex strongly capped and inhibited elongation. These results suggest that AIP has an active role in filament severing or depolymerization and that ADF/cofilin and AIP1 are distinct from gelsolin in modulating filament elongation.     

ADF/cofilin proteins translocate to mitochondria during apoptosis but are not generally required for cell death signaling

Non-muscle cofilin (n-cofilin) is a member of the ADF/cofilin family of actin depolymerizing proteins. Recent studies reported a mitochondrial translocation of n-cofilin during apoptosis. As these studies also revealed impaired cytochrome c release and a block in apoptosis upon small interfering RNA-mediated n-cofilin knockdown, n-cofilin was postulated to be essential for apoptosis induction. To elucidate the general importance of ADF/cofilin activity for apoptosis, we exposed mouse embryonic fibroblasts deficient for n-cofilin, ADF (actin depolymerizing factor), or all ADF/cofilin isoforms to well-characterized apoptosis inducers. Cytochrome c release, caspase-3 activation, and apoptotic chromatin condensation were unchanged in all mutant fibroblasts. Thus, we conclude that ADF/cofilin activity is not generally required for induction or progression of apoptosis in mammalian cells. Interestingly, mitochondrial association of ADF and n-cofilin during apoptosis was preceded by, and dependent on, actin that translocated by a yet unknown mechanism to mitochondria during cell death.     

Identification of yeast cofilin residues specific for actin monomer and PIP2 binding.

Cofilin/ADF is a ubiquitous actin-binding protein that is important for rapid actin dynamics in vivo. The long alpha-helix (helix 3 in yeast cofilin) forms the most highly conserved region in cofilin/ADF proteins, and residues in the NH2-terminal half of this alpha-helix have been shown to be essential for actin binding in cofilin/ADF. Recent studies also suggested that the basic residues in the COOH-terminal half of this alpha-helix would play an important role in F-actin binding. In contrast to these studies, we show here that the charged residues in the COOH-terminal half of helix 3 are not important for actin filament binding in yeast cofilin. Mutations in these residues, however, result in a small defect in actin monomer interactions. We also show that yeast cofilin can differentiate between various phosphatidylinositides, and mapped the PI(4,5)P2 binding site by using a collection of cofilin mutants. The PI(4,5)P2 binding site of yeast cofilin is a large positively charged surface that consists of residues in helix 3 as well as residues in other parts of the cofilin molecule. This suggests that cofilin/ADF proteins probably interact simultaneously with more than one PI(4,5)P2 molecule. The PI(4,5)P2-binding site overlaps with areas that are important for F-actin binding, explaining why the actin-related activities of cofilin/ADF are inhibited by PI(4,5)P2. The biological roles of actin and PI(4,5)P2 interactions of cofilin are discussed in light of phenotypes of specific yeast strains carrying mutations in residues that are important for actin and PI(4,5)P2 binding.     

植物肌动蛋白解聚因子ADF的研究进展

肌动蛋白解聚因子/丝切蛋白(actin depolymerizing factor,ADF/cofilin)是一种重要的肌动蛋白结合蛋白。在植物细胞中,ADF/cofilin通过与肌动蛋白相结合,在植物生长发育以及响应外界刺激方面起着重要的作用,以此对各种动态生命活动进行调控。该文对国内外近年来有关ADF/cofilin家族的序列结构特征及定位,与肌动蛋白的互作机制、促进细胞生长、抗生物和非生物逆境胁迫能力等的生物学功能,以及磷酸化作用、环境pH、PIP2对其功能影响的调控模式和作用机制进行了综述,为ADF/cofilin新的抗逆功能机制解析提供参考。     

肌动蛋白解聚因子/cofilin功能的研究进展

肌动蛋白解聚因子（actin depolymerizing factor,ADF）/cofilin家族是一类肌动蛋白结合蛋白,它们通过切断肌动蛋白纤丝并结合到肌动蛋白单体上,在重塑肌动蛋白骨架中发挥重要作用。就ADF/cofilin家族的结构特点、调控肌动蛋白动力学的机制及其功能的最新研究进展做一简要综述,并指出了目前在ADF/cofilin功能研究方面的不足和尚需解决的问题。     

The Effects of ADF/Cofilin and Profilin on the Conformation of the ATP-Binding Cleft of Monomeric Actin

Actin depolymerizing factor (ADF)/cofilin and profilin are small actin-binding proteins, which have central roles in cytoskeletal dynamics in all eukaryotes. When bound to an actin monomer, ADF/cofilins inhibit the nucleotide exchange, whereas most profilins accelerate the nucleotide exchange on actin monomers. In this study the effects of ADF/cofilin and profilin on the accessibility of the actin monomer''s ATP-binding pocket was investigated by a fluorescence spectroscopic method. The fluorescence of the actin bound ɛ-ATP was quenched with a neutral quencher (acrylamide) in steady-state and time dependent experiments, and the data were analyzed with a complex form of the Stern-Volmer equation. The experiments revealed that in the presence of ADF/cofilin the accessibility of the bound ɛ-ATP decreased, indicating a closed and more compact ATP-binding pocket induced by the binding of ADF/cofilin. In the presence of profilin the accessibility of the bound ɛ-ATP increased, indicating a more open and approachable protein matrix around the ATP-binding pocket. The results of the fluorescence quenching experiments support a structural mechanism regarding the regulation of the nucleotide exchange on actin monomers by ADF/cofilin and profilin.     

Determining the differences in actin binding by human ADF and cofilin.

The actin-depolymerizing factor (ADF)/cofilin family of proteins play an essential role in actin dynamics and cytoskeletal re-organization. Human tissues express two isoforms in the same cells, ADF and cofilin, and these two proteins are more than 70% identical in amino acid sequence. We show that ADF is a much more potent actin-depolymerizing agent than cofilin: the maximum level of depolymerization at pH 8 by ADF is about 20 microM compared to 5 microM for cofilin, but little depolymerization occurs at pH 6.5 with either protein. However, we find little difference between the two proteins in their binding to filaments, their severing activities or their activation of subunit release from the pointed ends of filaments. Likewise, they show no significant differences in their affinities for monomeric actin: both bind 15-fold more tightly to actin.ADP than to actin.ATP. Complexes between actin.ADP and ADF or cofilin associate with both barbed and pointed ends of filaments at similar rates (close to those of actin.ATP and much higher than those of actin.ADP). This explains why high concentrations of both proteins reverse the activation of subunit release at pointed ends. The major difference between the two proteins is that the nucleating activity of cofilin-actin.ADP complexes is twice that of ADF-actin.ADP complexes and this, in turn, is twice that of actin.ATP alone. It is this weaker nucleating potential of ADF-actin.ADP that accounts for the much higher steady-state depolymerizing activity. The pH-sensitivity is due to the nucleating activity of complexes being greater at pH 6.5 than at pH 8. Sequence analysis of mammalian and avian isoforms shows a consistent pattern of charge differences in regions of the protein associated with F-actin-binding that may account for the differences in activity between ADF and cofilin.     

Ins and outs of ADF/cofilin activity and regulation

The actin-binding proteins of the actin-depolymerisation factor (ADF)/cofilin family were first described more than three decades ago, but research on these proteins still occupies a front role in the actin and cell migration field. Moreover, cofilin activity is implicated in the malignant, invasive properties of cancer cells. The effects of ADF/cofilins on actin dynamics are diverse and their regulation is complex. In stimulated cells, multiple signalling pathways can be initiated resulting in different activation/deactivation switches that control ADF/cofilin activity. The output of this entire regulatory system, in combination with spatial and temporal segregation of the activation mechanisms, underlies the contribution of ADF/cofilins to various cell migration/invasion phenotypes. In this framework, we describe current views on how ADF/cofilins function in migrating and invading cells.     

Putting a new twist on actin: ADF/cofilins modulate actin dynamics.

The actin-depolymerizing factor (ADF)/cofilins are a family of essential actin regulatory proteins, ubiquitous among eukaryotes, that enhance the turnover of actin by regulating the rate constants of polymerization and depolymerization at filament ends, changing the twist of the filament and severing actin filaments. Genetic and cell-biological studies have shown that an ADF/cofilin is required to drive the high turnover of the actin cytoskeleton observed in vivo. The activity of ADF/cofilin is regulated by a variety of mechanisms, including specific phosphorylation and dephosphorylation. This review addresses aspects of ADF/cofilin structure, dynamics, regulation and function.     

Solution structure and dynamics of ADF from Toxoplasma gondii

Toxoplasma gondii ADF (TgADF) belongs to a functional subtype characterized by strong G-actin sequestering activity and low F-actin severing activity. Among the characterized ADF/cofilin proteins, TgADF has the shortest length and is missing a C-terminal helix implicated in F-actin binding. In order to understand its characteristic properties, we have determined the solution structure of TgADF and studied its backbone dynamics from ¹⁵N-relaxation measurements. TgADF has conserved ADF/cofilin fold consisting of a central mixed β-sheet comprised of six β-strands that are partially surrounded by three α-helices and a C-terminal helical turn. The high G-actin sequestering activity of TgADF relies on highly structurally and dynamically optimized interactions between G-actin and G-actin binding surface of TgADF. The equilibrium dissociation constant for TgADF and rabbit muscle G-actin was 23.81 nM, as measured by ITC, which reflects very strong affinity of TgADF and G-actin interactions. The F-actin binding site of TgADF is partially formed, with a shortened F-loop that does not project out of the ellipsoid structure and a C-terminal helical turn in place of the C-terminal helix α4. Yet, it is more rigid than the F-actin binding site of Leishmania donovani cofilin. Experimental observations and structural features do not support the interaction of PIP2 with TgADF, and PIP2 does not affect the interaction of TgADF with G-actin. Overall, this study suggests that conformational flexibility of G-actin binding sites enhances the affinity of TgADF for G-actin, while conformational rigidity of F-actin binding sites of conventional ADF/cofilins is necessary for stable binding to F-actin.     

Structure of the actin-depolymerizing factor homology domain in complex with actin

Actin dynamics provide the driving force for many cellular processes including motility and endocytosis. Among the central cytoskeletal regulators are actin-depolymerizing factor (ADF)/cofilin, which depolymerizes actin filaments, and twinfilin, which sequesters actin monomers and caps filament barbed ends. Both interact with actin through an ADF homology (ADF-H) domain, which is also found in several other actin-binding proteins. However, in the absence of an atomic structure for the ADF-H domain in complex with actin, the mechanism by which these proteins interact with actin has remained unknown. Here, we present the crystal structure of twinfilin's C-terminal ADF-H domain in complex with an actin monomer. This domain binds between actin subdomains 1 and 3 through an interface that is conserved among ADF-H domain proteins. Based on this structure, we suggest a mechanism by which ADF/cofilin and twinfilin inhibit nucleotide exchange of actin monomers and present a model for how ADF/cofilin induces filament depolymerization by weakening intrafilament interactions.     

The ADF/cofilin family: actin-remodeling proteins

The ADF/cofilins are a family of actin-binding proteins expressed in all eukaryotic cells so far examined. Members of this family remodel the actin cytoskeleton, for example during cytokinesis, when the actin-rich contractile ring shrinks as it contracts through the interaction of ADF/cofilins with both monomeric and filamentous actin. The depolymerizing activity is twofold: ADF/cofilins sever actin filaments and also increase the rate at which monomers leave the filament's pointed end. The three-dimensional structure of ADF/cofilins is similar to a fold in members of the gelsolin family of actin-binding proteins in which this fold is typically repeated three or six times; although both families bind polyphosphoinositide lipids and actin in a pH-dependent manner, they share no obvious sequence similarity. Plants and animals have multiple ADF/cofilin genes, belonging in vertebrates to two types, ADF and cofilins. Other eukaryotes (such as yeast, Acanthamoeba and slime moulds) have a single ADF/cofilin gene. Phylogenetic analysis of the ADF/cofilins reveals that, with few exceptions, their relationships reflect conventional views of the relationships between the major groups of organisms.